The Effect Of PRL-3 Regulated PI3K/Akt/mTOR Signal Pathway On The Death Of Myeloid Leukemia Cell And Its Mechanism

Objective 1 Screen the expression of PRL-3 and m TOR in different myeloid leukemia cell lines.2 Construct different lentiviral vectors to study the effect of PRL-3expression changes on the PI3K/Akt/m TOR signaling pathway in myeloid leukemia cells.3 To study the effect of PRL-3 inhibitor down-regulating the expression of PRL-3on the biological activity of myeloid leukemia cells and the PI3K/Akt/m TOR signaling pathway.4 To study the effect of m TOR inhibitor down-regulation of m TOR expression on the death of myeloid leukemia cells.5 To explore whether the combined application of PRL-3 inhibitors and m TOR inhibitors can synergistically promote the death of myeloid leukemia cells and the possible mechanism.Methods 1 The expression of PRL-3 and m TOR m RNA and protein in 3 different myeloid leukemia cell lines were detected by q PCR and Western blot.2 Construct specific expression lentiviral vector targeting PRL-3,K562,U937 cells were used as experimental cell line,we used puromycin to select stable transfection cell lines,screening of the effect of lentiviral transfection and the expression of m TOR by q PCR and Western blot.The changes in PI3K/Akt/m TOR signaling pathway and autophagy protein were detected by Western blot.3 After the PRL-3 inhibitor Pentamidine acted on K562 and U937 cells respectively,the experimental concentration of Pentamidine was determined by MTS assay and the expression changes of PRL-3 and m TOR were detected by q PCR and Western blot.DNA ploidy assay,Annexin V-PE/7-AAD double staining assay and Western blot were used to detect changes of biological activity indicator,including cell cycle distribution,apoptosis and autophagy.Western blot was used to detect changes in the expression of PI3K/Akt/m TOR signaling pathway proteins Akt,m TOR and their phosphorylated proteins.4 After the m TOR inhibitor Rapamycin acted on K562 and U937 cells respectively,the apoptosis and autophagy were observed by transmission electron microscope.5 After combined application of Rapamycin and Pentamidine on K562 and U937 cells respectively,Western blot was used to detect the expression changes of apoptosis and autophagy-related proteins such as Bcl2,Bax,LC3B,and to explore the possible mechanism of the simultaneous inhibition of the expression of m TOR and PRL-3 to promote the death of K562 and U937 cell.Results 1 In the three myeloid leukemia cell lines,the m RNA and protein expression of PRL-3 were higher degree in K562 cells,moderate degree in Kasumi-1cells and lower degree in U937 cells;the m RNA and protein expression of m TOR m RNA and protein were higher degree in K562 cells,moderate degree in Kasumi-1cells and lower degree in U937 cells.2 Successfully constructed a specific sh RNA lentiviral vector targeting PRL-3 and a PRL-3 overexpression lentiviral vector,respectively,which can infect K562 and U937 cells and stable expression GFP,then stable transfection was selected by puromycin,the best target for interference that detected by q PCR was MOI=20+enhanced infection In liquid P liquid,which could successfully down-regulate m RNA and protein of PRL-3 in K562 cells,and over-express m RNA and protein of PRL-3 in U937 cells;down-regulated of PRL-3,the m RNA and protein expression of m TOR in K562-KD group were down-regulated（P<0.05）.Over-expression of PRL-3,the m RNA and protein expression of m TOR in U937-OE group were up-regulated（P<0.05）.Western blot results showed that:compared with the control group,the expression of Akt protein in the K562-KD group did not change significantly（P>0.05）,the expression of p-Akt,m TOR and p-m TOR proteins were all decreased significantly（P<0.05）,the expression of autophagy protein ATG5 was increased significantly（P<0.05）,the ratio of LC3Ⅱ/LC3Ⅰwas decreased significantly（P<0.05）,and the expression of Beclin-1 protein did not change significantly（P>0.05）;compared with the control group,the expression of Akt protein in the U937-OE group did not change significantly（P>0.05）,the expression of p-Akt,m TOR,p-m TOR protein were increased significantly（P<0.05）,the expression of autophagy protein ATG5,Beclin-1 did not change significantly（P>0.05）,the ratio of LC3Ⅱ/LC3Ⅰwas increased significantly（P<0.01）.3 After the PRL-3 inhibitor Pentamidine acted on K562 and U937 cells respectively,the results of MTS assay showed that the experimental concentrations of the two cell lines were 4.960 n M and3.838 n M;the m RNA and protein expression of PRL-3 and m TOR were significantly down-regulated（P<0.05）.Compared with the respective non-administered groups:the results of MTS assay showed that the growth of K562 and U937 cells was inhibited（P<0.05）;the results of flow cytometry cycle and apoptosis test showed that the proportion of K562 and U937 cells in the G₂/M phase were increased significantly（P<0.05）,the proportion of K562 and U937 cells in the S phase were reduced significantly（P<0.05）and the total apoptosis rate was increased significantly（P<0.05）;the results of Western blot of autophagy protein showed that the expression of ATG5 in K562 and U937 cells were increased significantly（P<0.05）the ratio of LC3Ⅱ/LC3Ⅰwas decreased significantly（P<0.05）and the expression of Beclin-1 protein did not change significantly（P<0.05）;the results of Western blot of signal pathway proteins showed that there was no significant change in the Akt protein of the K562,U937 cells（P>0.05）;the expression of p-Akt,m TOR,and p-m TOR proteins were decreased significantly between K562 and U937 cells（P<0.05）.4 After the action of the m TOR inhibitor Rapamycin,transmission electron microscopy results showed that obvious nuclear pyknosis and apoptotic bodies were observed in K562 and U937 cells,but autophagosomes were not observed.After Rapamycin combined with Pentamidine acted on K562 and U937 cells respectively,the results of Western blot showed:compared with the respective Rapamycin-treated groups,the expression of Bcl2 protein in K562and U937 cells did not change significantly（P>0.05）,the expression of Bax protein and the ratios of Bax/Bcl2 in K562 and U937 cells were all increased significantly（P<0.01）,the ratios of LC3Ⅱ/LC3Ⅰwas decreased significantly（P<0.01）.Conclusion 1 In terms of m RNA and protein levels,the expression trends of PRL-3 and m TOR in different myeloid leukemia cell lines are consistent but the expression levels are different;it is determined that K562 cells with higher expression of both and U937 cells with lower expression of both are experimental cells.2Successfully constructed specific sh RNA lentiviral vectors targeting PRL-3 and PRL-3over-expression lentiviral vectors with stable down-regulation of PRL-3 in K562 cells and up-regulation of PRL-3 in U937 cells were selected.3 The expression of m TOR m RNA and protein will decrease with the down-regulation of PRL-3.On the contrary,the expression of m TOR will increase with the over-expression of PRL-3.4 Knockdown of PRL-3 in K562 cells can inhibit the PI3K/Akt/m TOR signaling pathway were inhibited by,down-regulate the expression of m TOR,and reduce the level of autophagy;over-expression of PRL-3 in U937 cells can activate the PI3K/Akt/m TOR signaling pathway and up-regulate the expression of m TOR.The increased expression of autophagy indicated that PRL-3 may affect autophagy by regulation of PI3K/Akt/m TOR signaling pathway.5 PRL-3 inhibitor Pentamidine can inhibit the PI3K/Akt/m TOR signaling pathway,inhibit cell proliferation,cycle arrest,cell apoptosis and reduce autophagy levels,which is consistent with conclusion 3.5 Single use of m TOR inhibitor Rapamycin can promote cell death and induce increased levels of autophagy;combined application of PRL-3 inhibitor Pentamidine,PRL-3 inhibitor can reduce the protective autophagy induced by Rapamycin,and synergistically promote the growth of myeloid leukemia cells death.